The goal of this research is to gain a comprehensive understanding of how the activities of the origin recognition complex (ORC) are regulated by interactions with DNA and accessory proteins. ORC is an evolutionarily conserved heteromeric protein complex whose activities are affected by chromosomal context. ORC is best known for its pivotal role in DNA replication where it marks chromosomal sites that serve as DNA replication origins. In addition to binding origins, ORC also binds to silencers that are also small DNA elements that depend on ORC for their function. However, silencers function inefficiently or not at all in DNA replication. Instead, silencers direct formation of repressive (silent) chromatin domains by nucleating the assembly of a specialized protein complex of SIR (silent information regulator) proteins that bind and modify nucleosomes. The focus of these studies has been one of four yeast silencers, HMR-E, a ~150 bp element necessary and sufficient to establish a ~4 kb silent chromatin domain at a locus in yeast called HMRa. In the last grant cycle, we defined the molecular and structural mechanisms governing an interaction between ORC and the specialized accessory protein Sir1 that defines ORC's role at silencers. We also learned that ORC binds HMR-E differently from several replication origins, and that these differences contribute to both HMR-E's positive role in forming silent chromatin and its virtual inability to function as a DNA replication origin. These and other data have raised new questions concerning the mechanisms that modulate the activities of ORC and SIR proteins in both chromatin structure and DNA replication. To address these questions we will: 1. Combine genetic and biochemical approaches to discern how the ORC-Sir1 interaction is regulated;2. Use biochemical and genetic approaches to define ORC-DNA complexes that differentially modulate ORC function;3. Use whole-genome approaches to define mechanisms that modulate ORC binding in vivo. The public health implications of understanding ORC activity are immense. ORC controls the premiere step in cell proliferation, a step essential for manipulating both normal (i.e., tissue regeneration) and uncontrolled (i.e., cancer) cell growth. Further, the ability to control ORC in species-specific manners will pave the way for developing inhibitors of the emerging class of fungal pathogens.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Molecular Genetics A Study Section (MGA)
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Carter, Anthony D
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University of Wisconsin Madison
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Hoggard, Timothy A; Chang, FuJung; Perry, Kelsey Rae et al. (2018) Yeast heterochromatin regulators Sir2 and Sir3 act directly at euchromatic DNA replication origins. PLoS Genet 14:e1007418
Kuznetsov, Vyacheslav I; Haws, Spencer A; Fox, Catherine A et al. (2018) General method for rapid purification of native chromatin fragments. J Biol Chem 293:12271-12282
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Hoggard, Timothy; Liachko, Ivan; Burt, Cassaundra et al. (2016) High Throughput Analyses of Budding Yeast ARSs Reveal New DNA Elements Capable of Conferring Centromere-Independent Plasmid Propagation. G3 (Bethesda) 6:993-1012
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Shor, Erika; Fox, Catherine A; Broach, James R (2013) The yeast environmental stress response regulates mutagenesis induced by proteotoxic stress. PLoS Genet 9:e1003680
Fox, Catherine A; Gartenberg, Marc R (2012) Palmitoylation in the nucleus: a little fat around the edges. Nucleus 3:251-5
Park, Sookhee; Patterson, Erin E; Cobb, Jenel et al. (2011) Palmitoylation controls the dynamics of budding-yeast heterochromatin via the telomere-binding protein Rif1. Proc Natl Acad Sci U S A 108:14572-7

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